Method of preparing stable sterile crystalline cephalosporins for parenteral administration

ABSTRACT

Sterile, essentially crystalline cephalosporins for parenteral administration are prepared by a freeze-drying process wherein, after rapidly cooling a solution of such cephalosporins to at least -15° C. in no more than 3 hours, the frozen solution is held between about &lt;0° C. and about -10° C. until the nucleation of the cephalosporin is substantially complete before subjecting said frozen solution to a high vacuum and a moderate amount of heat to sublime the water therefrom.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention is directed to an improved freeze-drying (lyophilization)process. More specifically the instant invention concerns afreeze-drying process wherein cephalosporins are prepared which aresterile, essentially crystalline and have good storage stability.

2. Prior Art

Freeze-drying is an old and often used process for removing a solventfrom a solute. While the process is cumbersome, expensive and slow, itprovides a method for removing a solvent without damaging heat labilesolutes. Antibiotics and other pharmaceuticals have been processed byfreeze-drying procedures for three or more decades and foods,particularly instant coffee, have been prepared by this method for manyyears. Ordinarily, a solution from which it is desired to recover thesolute in a relatively solvent-free state is frozen solid and thensubjected to an environment of a high vacuum and the temperature of theenvironment is raised to provide the units of heat absorbed in thesublimation of the solvent from the frozen solution. The temperature ofthe environment is kept below that which would result in the meltdown ofthe frozen solution. In practice, the temperature of the environment iscoordinated with the vacuum to produce the highest reasonablesublimation rate, avoiding a melting of the frozen mass.

Water is the solvent generally utilized in a freeze-drying process.Other solvents can be employed but are limited to those which becomesolid in the range of temperatures which can be practically employed inthe process and which will sublime under vacuum.

Although all of the material does not have to be in solution toeffectively operate a freeze-drying process, instant coffee being oneprobable example, this invention is concerned with a process whereincrystalline material is prepared in a freeze-drying procedure from atrue solution. In freeze-drying antibiotics and other pharamaceuticalsit has been the practice to follow the classic process outlined above;to wit, prepare solution, freeze to solid, subject to high vacuum, addheat, sublime solvent. However, when such a conventional procedure isfollowed, most cephalosporins come out as amorphous material. A statethat is undesirable because, generally, the amorphous powder is notstorage stable, even under refrigerated conditions. The basically whiteamorphous cephalosporin powders quickly begin to deteriorate to adisagreeable yellow. The yellow slowly appears and becomes noticeable inbetween 4 and 6 weeks at 4° C., and after 6 months at this temperaturegenerally is universal throughout the powder.

The cephalosporins involved in this invention can be recovered fromorganic solvents, such as ethanol in an essentially crystalline state.These crystals are equally as stable as the crystals prepared by thefreeze-drying process of the instant invention

However, recovering crystals of the cephalosporins for use in sterileampoule preparations for parenteral administration poses other problemsand conditions which are both inefficient, difficult and costly. Forexample, there is no effective way known to sterilize the crystals ofcephalosporins recovered from organic solvents so the entirecrystallization process must be carried out in an aseptic environment.In the large and extensive process required to sterilely crystallize thecephalosporins there are many opportunities for the admittance offoreign materials into the crystals which later on will show up assuspended material in a reconstituted ampoule of the antibiotic. No onehas yet developed an apparatus for filling dry material into an ampoulewhich will measure the material going into each ampoule with as good aconsistency and precision as can be routinely achieved with liquidfilling equipment.

Accordingly, it is an object of this invention to provide a process offreeze drying cephalosporing, selected from the group consisting ofcephalothin sodium, cephaloridine betaine, and cefazolin sodium thatwill result in sterile essentially crystalline cephalosporins forparenteral administration.

Another object of this invention is to provide a process which willinclude the sterile liquid filling of a measured volume of a sterilesolution of a known concentration of a cephalosporin into an ampoulewherein such cehalosporin is recovered from such solution as anessentially crystalline material for parenteral administration which isstorage stable.

Still another object of this invention is to provide an ampoulecontaining an essentially crystalline cephalosporin which is storagestable and which upon reconstitution for parenteral administration issubstantially free of foreign suspended material.

SUMMARY

Now it has been discovered that a storage stable, sterile, essentiallycrystalline cephalosporin selected from the group consisting ofcephalothin sodium, cephaloridine betaine and cefazolin sodium forreconstitution for parenteral administration can be prepared by afreeze-drying procedure comprising the following steps: (a) Thecephalosporin is dissolved in water in a concentration of between about15 and about 40 percent. (b) The cephalosporin preparation from (a) issterile filtered into a previously sterilized container. (c) Thecephalosporin preparation from (b) is rapidly lowered to a temperaturebelow at least -15° C. within an interval not longer than 3 hours. (d)The temperature of the cephalosporin preparation from (c) is raised tobetween <0° C. and about -10° C. (e) The cephalosporin preparation from(d) is held between <0° C. and about -10° C. until the nucleation of thecrystals of said cephalosporin is substantially complete. (f) Thecephalosporin preparation from (e) is subjected to an environment inwhich the pressure is maintained at a maximum of lmm absolute. And, (g)the temperature of the environment in which the cephalosporinpreparation is maintained at a maximum of lmm absolute is raised to 50°C. or below, subliming the water from the cephalosporin preparationresulting in the recovery of an essentially crystalline cephalosporinhaving a moisture content of not more than 6.0 percent.

DESCRIPTION OF THE PREPARED EMBODIMENTS

The useful process of the present invention comprises a procedureutilizing a freeze-drying operation wherein an aqueous preparation of acephalosporin selected from the group consisting of cephalothin sodium,cephaloridine betaine and cefazolin sodium is subjected to a temperaturewhich will rapidly, within a period of no longer than 3 hours, lower thetemperature of such preparation to at least -15° C., preferably about-20° C. At this temperature dendritic crystals of ice begin to form inwhich there is little or no cephalosporin entrained. Then thetemperature of the environment in which the cephlosporin preparation isheld is raised to between <0° C. and -10° C. The rise in the temperatureestablishes an environment which is favorable for an increase in thesize of the dendritic ice crystals, and the nucleation of thecephalosporin crystals begins.

After the temperature has been raised to between <0° C. and -10° C. thecephalosporin preparation is held at such a temperature for from about 2to about 48 hours to provide a time interval during which substantiallyall of the cephalosporin solute is nucleated and essentially all of theliquid water becomes ice in which little or no cephalosporin remainsentrained therein. The time required for the nucleation of the variouscephalosporins varies. For example, between 12 and 24 hours of exposureto a temperature between <0° C. and -10° C. is required to nucleatesubstantially all of the cephalothin sodium contained in a cephaloporinpreparation undergoing freeze drying. From about 12 to about 48 hoursare required to nucleate all of the cephalosporin betaine from acephalosporin preparation, while a cefazolin sodium nucleation will besubstantially complete in between about 2 and about 8 hours; bothbetween <0° C. and about -10° C.

Following the critical steps just described, a conventionalfreeze-drying operation is employed to sublime the ice leaving sterilecephalosporin crystals having a moisture content of no more than 6percent. Such crystals have a suitable storage stability; three years ormore at room temperature, and are appropriate for reconstitution forparental administration.

The crux of the instant invention is the establishment of conditionswhich are favorable to and result in the development of dendritic icecrystals rather than crystals having a fine platelet or hexagonalstructure. The latter two structures are generally developed by slowcooling. Such structures entrain the cephalosporin therein and in afreeze-drying procedure the cephalosporins are deposited in an amorphousstate as the ice is sublimed.

In one aspect of the present invention a sterile, essentiallycrystalline cephalosporin selected from the group consisting ofcephalothin sodium, cephaloridine betaine and cefazolin sodium forreconstitution for parenteral administration is prepared by a methodcomprising the following steps: (a) The cephalosporin is dissolved inwater. (b) The aqueous cephalosporin preparation from (a) is filteredthrough a sterilizing filter into a previously sterilized container. (c)The preparation from (b) is cooled rapidly to a temperature below atleast -15° C. within a period of not more than 3 hours. (d) Thepreparation from (c) is warmed to a temperature between <0° C. and about-10° C. (e) The preparation from (d) is held at a temperature between<0° C. and -10° C. until the nucleation of the crystals of saidcephalosporin is substantially complete. (f) The preparation from (e) issubjected to an environment wherein the pressure is maintained at amaximum of no more than lmm absolute. (g) The temperature of theenvironment to which the preparation from (f) is exposed is raised to amaximum of about 50° C., avoiding the melting of such preparation. And,(h) the ice is sublimed from the preparation from (g) until theresulting crystals of said cephalosporin have a moisture content of nomore than 6 percent.

Any cephalosporin material, of those included in this invention, whichis of a pharmaceutical grade can be dissolved in water to provide theaqueous solution used in the useful process detailed herein. Aconcentration of the cephalosporin between about 15 and about 40 percent(W/V) is appropriate. Preferably, concentrations of cephalothin sodiumbetween about 15 and about 33 percent (W/V); of cephaloridine betainebetween about 20 and about 33 percent (W/V); and of cefazolin sodiumbetween about 20 and about 40 percent (W/V) are satisfactory for use indeveloping the dendritic crystals of ice which are formed when theconditions which are detailed herein, as those required in practicingthis invention, are followed.

The sterilization of the aqueous solutions of cephalosporins can beachieved by filtering such solutions through sterile filtering meansknown to those skilled in the art and collecting the filtrate in apreviously sterilized container. Illustratively, sterile filtering canbe effected using a heat sterilized plate and frame filter pressequipped with an asbestos pad, or a filtering membrane of celluloseacetate or or nitrate, or a candle having a porosity below 0.22 μm.

The dendritic ice crystals, the formation of which is an essentialelement of this invention, are achieved by rapidly cooling the sterilecephalosporin preparation to a temperature at least below -15° C. It wasfound that when this temperature was effected within a 3 hour periodfrom the time the cooling was commenced, the formation of dendriticrather than platelet or hexagon crystals was assured. It was also foundthat cooling the cephalosporin preparation to a temperature below -20°C. was of no additional benefit, but it is essential that the -15° C. bereached throughout the cephalosporin preparation. To accomplish thisneed it is required that the temperature of the environment in which thecephalosporin preparation is cooled to at least -15° C. be considerablylower than the -15° C. to achieve this temperature within the 3 hourperiod. Moreover, the actual size of the individual volume of thepreparation to be reduced to the -15° C. level will influence the actualoperating temperature to which the preparation is exposed. For example,if the volume of the preparation is a relatively large mass in thefrozen state, a substantially lower environment temperature will berequired in order to lower the temperature throughout the frozen mass to-15° C. than will be required if the individual volumes are relativelysmall, such as the volume of a 20 percent solution required in a ampouleto result in a 1g quantity of crystalline cephalosporin being depositedtherein at the conclusion of the subliming operation, describedhereinafter.

In actual practice it is sometimes the custom to provide a thermocouplein a location approximating the center of the frozen mass to indicatethe temperature at that point in such mass. If the temperature at suchpoint is not dropping sufficiently fast to reach the -15° C. temperaturewithin 3 hours, additional refrigeration can be added to accomplish therequired temperature in the specified time.

Once the -15° C. temperature has been achieved, the physical conditionsconducive to the development and propagation of dentritic ice crystalshave been established. At this temperature the nucleation of the variouscephalosporin crystals will proceed at different rates. In any event,though, nucleation is slower at lower temperatures. To advance the rateof nucleation the temperature is raised to a level that is conducive tothe growth of the dendritic crystals and a reasonable rate of nucleationof the cephalosporin crystals. However, the temperature must not beraised to a point where the frozen mass will begin to melt. Therefore, atemperature range of between <0° C. and about -10° C. was found to beeffective for completing the crystal nucleation. A temperature of about-8° C. was found to be especially suitable for the cephalosporinsinvolved in this invention. The symbol < used throughout thisspecification means below; for example, <0° C. means below zero Celsius;i.e., cold enough to maintain the cephalosporin preparation in thefrozen state.

The frozen mass of cephalosporin preparation is held at between <0° C.and -10° C., preferably about -8° C. for between about 2 and about 48hours. It was found that the nucleation of cephalothin sodium crystalswas essentially complete between about 12 and about 24 hours from anaqueous concentration of between about 15 and about 33 percent when thefrozen mass was held between <0° C. and -10° C. Cephaloridine betainecrystals were essentially, completely nucleated from an aqueousconcentration of between about 20 and about 40 percent when a frozenmass of such a preparation was held between <0° C. and -10° C. forbetween 12 and 48 hours. Cefazolin sodium nucleates much faster atbetween <0° C. and -10° C., and was essentially complete from an aqueousconcentration between about 15 and about 40 percent in between about 2and about 8 hours.

After the nucleation of the cephalosporin crystals is substantiallycomplete, a conventional freeze-drying operation is utilized to sublimethe ice from the frozen mass leaving a deposit of sterile essentiallycrystalline cephalosporin.

The cephalosporin preparation wherein the nucleation of the crystals issubstantially complete is subjected to an environment where the pressurecan be reduced to a practical maximum of no more than lmm mercuryabsolute. It is preferable to reduce the pressure much more than to almm absolute. The best results are obtained with an absolute pressure ofbetween about 0.05mm and about 0.2mm. This latter pressure range isordinarily readily attainable in both laboratory and commercialfreeze-drying apparatus, the design, construction and operation of whichare all well known to those skilled in the art. After the pressure ofthe environment described above has been reduced to an operating level,heat is introduced into such an environment. The temperature of theenvironment is raised to a point where the maximum sublimation rate canbe achieved without melting the frozen mass. As a general rule, thetemperature and the pressure are inversely related; the more effectivethe pressure reduction, the higher the temperature which can be employedin the subliming operation. As a common guide it can be said that amaximum environment temperature of 50° C. can be reached with a highlyefficient vacuum system where the absolute pressure is maintained atabout 0.05mm absolute (50μm). In any event, the temperature should beraised slowly so as to avoid overloading the pressure-reducing systemwhich can produce an undesirable melting of the frozen mass. Preferably,the temperature of the environment in the subliming operation should bemaintained between from 20° C. and about 40° C. with the pressure heldat or below 0.2mm absolute.

Subliming of the ice from the frozen mass is continued until themoisture content of the cephalosporin crystals is below 6 percent.Cephalothin sodium crystals are not hydrated, and it is essential tocontinue the sublimation until the moisture content is below 1 percent.Such a specification assures physical stability of the resultingcrystals. Cephaloridine betaine crystallizes with about one mole ofwater; and, consequently, the final moisture content of the crystalsshould be held below about 3.5 percent, which includes the water ofhydration. Cefazolin sodium crystallizes as the pentahydrate. Holdingthe crystals at 25° C-30° C., after the ice has been completelysublimed, converts the penta- to the hemi-hydrate. Such crystals cancontain up to about 6 percent moisture.

The cephalosporins prepared as detailed above are essentiallycrystalline. For example, physical analyses of cephalothin sodiumindicated a crystallinity of between about 92 and about 98 percent,cephaloridine betaine was practically 100 percent, and cefazolin sodiumhemi-hydrated was greater than 90 percent. In any event, a sufficientlyhigh amount of crystallinity was obtained to impart storage stability;i.e., an absence of a yellowing of the substance, and loss ofmicrobiological potency for up to 3 years at room temperature. Thesecephalosporin crystals can be sterile filled into previously sterilizedampoules in appropriate quantities for reconstitution for parenteraladministration. The three cephalosporins which are involved in theinstant invention are all in use today for combatting susceptiblepathological organisms in sick people.

In another aspect of this invention the procedure outlined and discussedin detail hereinbefore is augmented by an additional step whichcomprises filling a measured volume of the sterile aqueous solution fromstep (b) into a previously sterilized ampoule, such measured volumecontaining the quantity of the cephalosporin which is desired in suchampoule after the freeze-drying operation. The ampoules containing thesterile aqueous solution of the cephalosporin are then processed in thesame manner as described above. The resulting freeze-dried cephalosporinampoule is ready for sterile stoppering and capping.

In practice it is preferred to sterile fill a measured volume of thesterile aqueous solution into a previously sterilized ampoule as atleast two beneficial results are obtained. First, a more precise andconsistent quantity of the cephalosporin can be filled into an ampoulein the liquid form than in the solid (crystals or powder) form. And,second, it is much easier to achieve and maintain sterile operatingconditions in liquid filling operations than in dry filling operations.Moreover, air pollution is less of a problem when handling liquids thandry materials.

What is claimed is:
 1. A method of preparing a sterile, essentiallycrystalline cephalosporin selected from the group consisting ofcephalothin sodium, cephaloridine betaine and cefazolin sodium forreconstitution for parenteral administration comprising the steps of:a.dissolving said cephalosporin in water; b. filtering the solution from(a) through a sterilizing filter; c. cooling the sterile filtrate from(b) rapidly to a temperture below at least -15° C. within an intervalnot longer than 3 hours; d. warming the preparation from (c) to between<0° C. and about -10° C.; e. maintaining the temperature of thepreparation from (d) between <0° C. and about -10° C. until thenucleation of the crystals of said cephalosporin is substantiallycomplete; f. reducing the pressure of the environment in which thepreparation from (e) is maintained to a maximum of lmm absolute; g.raising the temperature of the environment in which the preparation from(f) is maintained to a maximum of about 50° C., avoiding the melting ofsuch preparation; and h. subliming the water from the preparation from(g) until the resulting crystals of said cephalosporin have a moisturecontent of no more than 6.0 percent.
 2. The method according to claim 1wherein the concentration of the cephalosporin in the aqueous solutionis between about 15 and about 40 percent (W/V).
 3. The method accordingto claim 2 wherein the concentration of cephalothin sodium in theaqueous solution is between about 15 and about 33 percent (W/V).
 4. Themethod according to claim 2 wherein the concentration of cephaloridinebetaine is between about 20 and about 40 percent (W/V).
 5. The methodaccording to claim 2 wherein the concentration of cefazolin sodium inthe aqueous solution is between about 15 and about 40 percent (W/V). 6.The method according to claim 1 wherein the preparation from step (d) ismaintained between <0° C. and about -10° C. for between about 2 to about48 hours.
 7. The method according to claim 6 wherein when thepreparation is cephalothin sodium such preparation is maintained between<0° C. and about -10° C. for between about 12 and about 24 hours.
 8. Themethod according to claim 6 wherein when the preparation iscephaloridine betaine such preparation is maintained between <0° C. andabout 3110° C. for between about 12 l and about 48 hours.
 9. The methodaccording to claim 6 wherein when the preparation is cefazolin sodiumsuch preparation is maintained between <0° C. and about -10° C. forbetween about 2 and about 8 hours.
 10. The method according to claim 1wherein the pressure is reduced to between about 0.05 and about 0.20mmof mercury absolute (50 to 200μm absolute) and the temperature of theenvironment is raised slowly to between about 0° C. and about 40° C.maintaining an absolute pressure of no more than 0.20 mm or mercuryavoiding the melting of said cephalosporin preparation.
 11. The methodaccording to claim 1 wherein the sublimation of the water from saidcephalosporin preparation is continued until the moisture content of theresulting crystals of said cephalosporin have a moisture content ofbetween about 0.1 and about 6.0 percent.